Biochemistry, Biophysics, and Structural Biology Commons^™

Development Of 31p Nuclear Magnetic Resonance Methods For The Study Of Phosphate Metabolisms In E. Coli And B. Subtilis, Fabian M. Dayrit, Emilyn Q. Espiritu, Noreen Gonzalez, Nina Rosario L. Rojas, Jennifer T. Aguilan, Antonio M. Basilio, Edward T. Chainani, Enrico Cruz, Bernadette Matanguihan

Environmental Science Faculty Publications

31P NMR experiments were performed on Escherichia coli and Bacillus subtilis at various temperatures under aerobic and anaerobic conditions. The total soluble intracellular phosphate concentration was estimated to be 2 x 10^-17 mole/cell, while intracellular orthophosphate concentration was around 1 x 10^-17 mole/cell. Addition of glucose resulted in a general decrease in intracellular pH and was accompanied by the formation of sugar monophosphates. The concentrations of soluble intracellular phosphates, both inorganic and organic phosphates, were estimated by integration versus methylene diphosphonic acid (MDPA) standard. Although intracellular and extracellular orthophosphate could be observed, these appear to exchange rapidly on the NMR …

Plasmd Bearing A Cdna Copy Of The Genome Of Bovine Viral Diarrhea Virus, Chimeric Derivatives Thereof, And Method Of Producing An Infectious Bovine Wral Darrheavirus Using Sad Plasmid, Ruben O. Donis, Ventzislav B. Vassilev

School of Veterinary and Biomedical Sciences: Faculty Publications

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a …

In Situ Hybridization For The Detection And Localization Of Swine Chlamydia Trachomatis, C. Chae, D.-S. Cheon, D. Kwon, O. Kim, B. Kim, J. Suh, D. G. Rogers, K. D. E. Everett, A. A. Anderson

School of Veterinary and Biomedical Sciences: Faculty Publications

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4–7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were …

Early And Late Endosomal Compartments Of Entamoeba Histolytica Are Enriched In Cysteine Proteases, Acid Phosphatase And Several Ras-Related Rab Gtpases, Lesly A. Temesvari, Ed Harris, Samuel L. Stanley Jr., James Cardelli

Department of Biochemistry: Faculty Publications

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding …

Affinity Chromatography: A Review Of Clinical Applications, David S. Hage

David Hage Publications

Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLCbased methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with …

Carmoviruses (Tombusviridae), Feng Qu, Thomas Jack Morris

School of Biological Sciences: Faculty Publications

Carmovirus is one of two officially recognized genera of Tombusviridae. Members of Tombusviridae all have icosahedral virions of about 30 nm in diameter with T = 3 symmetry that consists of 180 coat protein (CP) subunits of about 38- 43 kDa and a single-stranded (ss) RNA genome ranging in size from 4.0 to 4.7 kb. Carmoviruses share recognizable sequence similarity in both the polymerase and structural genes with members of the other genus, Tombusvirus. Sequence comparisons and phylogenetic analysis of these genes support the view that the genera are distinct but closely related. In addition, the genome organization of the …

Investigating The Allosteric Behavior Of Malate Dehydrogenase From Escherichia Coli, Eman M. Ghanem

Masters Theses

Regulatory mechanisms of malate dehydrogenase from E.coli (eMDH) involving NADH as an allosteric effector were investigated. The reaction was studied in both directions: malate oxidation and oxaloacetate reduction. When malate was the variable substrate, a plot of rate against substrate concentration was sigmoidal in the presence of 0.065 mM NADH, which indicates the presence of an allosteric site for NADH on the enzyme. Binding of NADH at the allosteric site causes conformational changes in the active site and, thereby, changes the catalytic activity of the enzyme. An increase in K_m value, from 1.3 to 3.9 mM malate was observed, …

Temporal And Spatial Variation In Abundance Of Migratory Birds At Kennesaw Mountain, Georgia, Andrew Allen Kinsey

College of Graduate Studies: Theses & Dissertations (1964–2006)

Migratory birds spend a significant part of each year in transition between their breeding and resting grounds. During this transitional period migrants rely on stopover sites to provide them with the necessary habitat to replenish lost energy stores, rest, and wait for favorable weather conditions before continuing migration. The majority of stopover work related to passerines in North America has occurred along coastal migration routes. Meanwhile, relatively little work has been conducted at inland stopover sites, areas which may be equally important to migrating birds. Kennesaw Mountain is an isolated geographically prominent peak located in the foothills of the southern …

Detection Of Ehrlichia Chaffeensis In Ticks Of The Georgia Coast And Barrier Islands Using Polymerase Chain Reaction, John Edward Whitlock

College of Graduate Studies: Theses & Dissertations (1964–2006)

Human monocytic ehrlichiosis (HME) is an emerging, zoonosis transmitted to mammals by ixodid ticks. Prevalence of infected ticks and distribution of infection foci indicate relative risk of human exposure to ehrlichiosis and may be influenced by factors such as geographic isolation and human disturbance. To test this, individual and pooled lone star ticks, Amblyomma americanum (L.), collected from 3 populations from the coast and barrier islands of Georgia were screened for Ehrlichia chaffeensis Anderson, Dawson, Jone, and Wilson, the agent of HME. A species-specific, nested polymerase chain reaction (PGR) assay was used to amplify a 572 base pair fragment of …

Phorbol 12-Myristate 13-Acetate Stimulates Lysophosphatidic Acid Secretion From Ovarian And Cervical Cancer Cells But Not From Breast Or Leukemia Cells, Zhongzhou Shen, Jerome Belinson, Richard E. Morton, Yan Xu

Chemistry Faculty Publications

Lysophosphatidic acid (LPA) is present in ascites from patients with ovarian cancer. It stimulates calcium release and growth of ovarian cancer cells bothin vitroandin vivo.Recently, we found that LPA levels were significantly elevated in plasma from patients with ovarian cancer and other gynecological cancers. In contrast, LPA levels were not elevated in patients with breast cancer and leukemias. In view of this, we investigated whether gynecological cancer cells could produce LPA. LPA was extracted from the supernatant of cells culturedin vitroand purified by thin layer chromatography. After hydrolysis and transmethylation, the fatty acid derivatives were analyzed by gas chromatography. We …

Principal Component Mapping Applied To Raman Microspectroscopy Of Fiber-Reinforced Polymer Composites, C. M. Stellman, K. S. Booksh, A. R. Muroski, M. P. Nelson, Michael L. Myrick

Faculty Publications

We describe the development and implementation of a spectroscopic Raman imaging system using Principal Component data analysis. Raman microspectroscopy followed by multivariate data analysis and principal component mapping has been used to investigate the spatial variations of chemical and physical properties at interfaces in glass-reinforced composites on the micrometer scale. A protocol for monitoring chemical and physical variances via multivariate statistics has been developed and a preliminary study of a "real world" glass-reinforced composite is presented. It is demonstrated that multivariate Raman imaging is more precisely interpretable than traditional univariate methods and that by appropriately choosing distinct spectral regions (such …

Biochemistry Of Hemolysin Toxin Activation By Fatty Acylation: Characterization Of An Internal Protein Acyltransferase, Michael S. Trent

Electronic Theses and Dissertations

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and …

Selenocysteine-Containing Proteins In Mammals, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Since the recent discovery of selenocysteine as the 21st amino acid in protein, the field of selenium biology has rapidly expanded. Twelve mammalian selenoproteins have been characterized to date and each contains selenocysteine that is incorporated in response to specific UGA code words. These selenoproteins have different cellular functions, but in those selenoproteins for which the function is known, selenocysteine is located at the active center. The presence of selenocysteine at critical sites in naturally occurring selenoproteins provides an explanation for the important role of selenium in human health and development. This review describes known mammalian selenoproteins and discusses recent …

Dynamics Of Biomolecules: Assignment Of Local Motions By Fluorescence Anisotropy Decay, Carl N. Bialik, Barnabas Wolf, Edward L. Rachofsky, J. B. A. Ross, William Laws

Chemistry and Biochemistry Faculty Publications

Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two …

Accumulation Of Methotrexate In Human Tissues Following High-Dose Methotrexate Therapy, M P. Iqbal

Department of Biological & Biomedical Sciences

Methotrexate concentration was analyzed in a number of tissues of a patient of osteogenic sarcoma who had been on high-dose methotrexate therapy for nearly 6 months. Gall bladder and kidney contained the highest concentration of the drug, followed by testis, small intestine, skeletal muscle, bone marrow, lung, spleen, heart and liver. Although, compared to kidney the liver contained relatively small amount of the drug, yet nearly 1/5th of the total drug in liver was in bound form. This bound form of methotrexate is most likely associated with multiple forms of dihydrofolate reductase. The total concentration of methotrexate in kidney is …

Monte Carlo Simulation Of An Ion-Dipole Mixture As A Model Of An Electrical Double Layer, Douglas Henderson, Dezso Boda, Kwong-Yu Chan

Faculty Publications

Canonical Monte Carlo simulations were performed for a nonprimitive model of an electrical double layer. The ions and the solvent molecules are modeled as charged and dipolar hard spheres, respectively, while the electrode as a hard, impenetrable wall carrying uniform surface charge. We found that the ion-dipole model gives a reasonable description of the double layer for partially charged ions with small to moderate dipole moments, or equivalently for an "effective" dielectric constant. Density, polarization and mean electrostatic potential profiles are reported. Strong layering structure, and at higher charges, charge inversion in the second layer were found. With appropriate choices …

Interagency Lake Mead And Las Vegas Wash Monitoring Program: Standard Operating Procedures Manual, Bureau Of Reclamation, City Of Henderson Water Reclamation Facility, City Of Las Vegas Water Pollution Control Facility, Clark County Sanitation District, Nevada, Southern Nevada Water Authority

Publications (WR)

A number of agencies sample Lake Mead and the Las Vegas Wash on a routine basis at several locations. In order to share and properly interpret the data, the Bureau of Reclamation, Southern Nevada Water Authority and the three Wastewater Treatment Facilities (City of Las Vegas, Clark County Sanitation District and City of Henderson) formed a committee to examine sampling and analytical protocols and to share information with the goal of maximizing the data quality. The group first met in April 1997. It was agreed that an effort should be made to discuss and compare specific sampling and analytical techniques …

Urinary N-Acetyl-Beta-D-Glucosaminidase In Rheumatoid Arthritis, M P. Iqbal, A A. Ali, M A. Waqar, N Mehboobali

Department of Biological & Biomedical Sciences

Excretion of urinary N-acetyl beta-D-glucosaminidase (NAG) and its isoenzyme patterns were studied in two groups of patients with rheumatoid arthritis (RA) and in normal control subjects. Urine samples were collected from 30 seropositive RA patients, 19 seronegative RA patients, and 15 normal healthy subjects. All the patients and normal subjects were assessed to have normal liver and kidney functions. A small portion of the urine sample was dialyzed against 0.01 M phosphate buffer, pH 7.0 and NAG activity was monitored. Mean +/- SD values of urinary NAG in seropositive RA patients, in seronegative RA patients and in normal healthy subjects …

Simultaneous Formation Of Functional Leading And Lagging Strand Holoenzyme Complexes On A Small, Defined Dna Substrate, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by …

Localization Of Sodium, Potassium-Adenosine Triphosphatase In The Cerebral Cortex Of Developing Rats, Maki Ujiie

Loma Linda University Electronic Theses, Dissertations & Projects

Active transport via Na⁺, K⁺-ATPase plays an important role in establishing and maintaining proper ionic gradient across all mammalian cell membranes. A strict regulation of Na⁺, and K⁺ ion levels is especially important in excitable cells such as neurons and myocytes. Inhibition of the enzyme is associated with several pathological conditions and has serious detrimental effects on cerebral cortical cells. Yet the enzyme appears to be limited in amount and distribution in newborn rats. In this study, Na_⁺, K⁺- ATPase expression and distribution in early postnatal rats were studied …

The Effects Of Exposure To Extremely Low-Amplitude, Low-Frequency Electric Fields On Skeletal Tissue, In Vitro, Robert James Fitzsimmons

Loma Linda University Electronic Theses, Dissertations & Projects

In vivo experiments have demonstrated (a) that transient electrical potentials are induced in impact-loaded bone, and (b) that bone formation is increased in response to electrical stimulation. Therefore, it has been hypothesized that, in response to mechanical loading, mechanical energy in bone is transduced into biologically relevant electrical energy. In order to study the mechanism through which electric fields may increase bone formation, a model system was developed were bone cells or bone organ cultures were exposed to a capacitively-coupled electric field of low frequency (8-25 Hz) and extremely low amplitude (estimated 10^-7 V/cm in the culture medium) for …

Study Of The Baie Gene In Bile Acid 7Α-Dehydroxylating Bacteria, Yasemin Kaya

Masters Theses & Specialist Projects

Intestinal bile acid 7α-dehydroxylating bacteria have recently been implicated in cholesterol allstone disease. Eubacterium sp. V.P.I. 12708, a bile acid 7α-dehydroxylating bacterium, contains multiple bile acid inducible (bai) genes which encode the enzymes responsible for bile acid 7a-dehydroxylation. The baiE gene encodes a bile acid dehydratase activity in Eubacterium sp.V.P.I. 12708. Using the polymerase chain reaction assay we determined the presence or absence of baiE-like genes in five clostridial bile-acid 7α-dehydroxylating strains: Clostridium sp. TO-931, Clostridium sp. HD-17, Clostridium sp. TN-271, Clostridium bifermentans 1-55, and Clostridium sordellii ATCC 9714. Results from all the strains tested showed amplification at the predicted …

In Vitro Assessment Of The Toxicity Of Cocaine And Its Metabolites In The Human Umbilical Artery, Tessa L. Long

Electronic Theses and Dissertations

An in vitro model was used to assess the effect of cocaine and its metabolites on the umbilical artery. Objectives were to pharmacologically confirm the presence of adrenergic innervation using tyramine, evaluate the ability of cocaine, benzoylecgonine, norcocaine and cocaethylene to potentiate vasoconstriction by serotonin and norepinephrine, examine the ability of ketanserin to block the enhanced vasoconstriction produced by cocaine, and determine displacement of 3 H-ketanserin by serotonin, norepinephrine, tyramine and mianserin. The vasoconstrictive effect of tyramine (100 μM) was enhanced in the presence of cocaine by 257%. Vasoconstrictive effects of serotonin and norepinephrine were significantly enhanced by cocaine by …

Expression, Isolation And Purification Of Human Zona Pellucida Protein 3, Ting-Fung Chi

Theses and Dissertations in Biomedical Sciences

Mammalian fertilization involves interactions of sperm surface receptors with the ligands of the zona pellucida, an extracellular matrix surrounding the ovulated oocytes. In humans, the zona pellucida is composed of three major glycoproteins. One of them, ZP3, participates in the primary sperm binding and in the subsequent triggering of the spermatozoa's acrosome reaction. Studies on the role of this specific protein in the human fertilization process are hampered by the limited amount of available biologically functional proteins.

By use of a pcDNA 3.1(+) expression vector, a transfecting-vector was constructed containing a 1.3 kb histidine tagged hZP3 cDNA. This histidine tagged …

Development Of Fluorescent Techniques In The Kinetic Characterization Of Reverse Transcriptase, Kevin Kevin Howerton

Chemistry & Biochemistry Theses & Dissertations

Reverse transcriptase (RT) is an RNA-directed DNA polymerase isolated from human immunodeficiency virus (HIV) and other retroviruses. It has been the primary target for anti-HIV research since the discovery of its vital role in the retroviral life-cycle. The conventional method for carrying out kinetic characterization and inhibition studies of reverse transcriptase involves the use of radioactive materials. Although this method is effective, it is both time consuming, due to a necessary repeated washing step, and hazardous to the worker and environment. Furthermore, strict laws limit the use of radioactive materials to licensed workers and laboratories. In response to these concerns, …

Characterization Of The Biological Functions Of Human Recombinant Zona Pellucida Protein 3, Yu Wen Juan

Theses and Dissertations in Biomedical Sciences

Recombinant human zona pellucida protein 3 (rhZP3), expressed. isolated and purified from PA-1 cells, is characterized for its biological activity and the role in the signal transduction pathway. Characterization of the biological activity of rhZP3 was detected by hemizona assay and immunofluorescence staining of acrosome reaction The results indicated that rhZP3 exhibited an inhibition in the binding assay (HZI 43.6 +/-3.3; n = 9; 30 ng/mL rhZP3) and induction of acrosome reaction (198.6% +/- 77.2% increase from baseline; n = 29; 30 ng/mL rhZP3). It was further confirmed by the transmission electron microscopy that there was no difference in morphology …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

The Snare Machinery Is Involved In Apical Plasma Membrane Trafficking In Mdck Cells, Seng Hui Low, Steven J. Chapin, Christian Wimmer, Sidney W. Whiteheart, László G. Kömüves, Keith E. Mostov, Thomas Weimbs

Molecular and Cellular Biochemistry Faculty Publications

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in …

Solute-Solvent Pair Distribution Functions In Highly Asymmetric Additive Hard Sphere Mixtures, Douglas Henderson, Kwong-Yu Chan

Faculty Publications

Contact values for the solute-solvent pair distribution function in an additive hard sphere mixture, as computed from the Henderson-Chan (HC) formulas, are compared with the recent Monte Carlo (MC) data and formula of Matyushov and Ladanyi (ML) [J. Chem. Phys. 107, 5815 (1997)]. The agreement is found to be excellent. The negative finding of ML is due to a misprint in one of the HC publications. We find the HC formula to be superior to the ML formula when compared to our MC data [Mol. Phys. 91, 1137 (1997)] for the case where the ratio of diameters is large and …

Transcriptional Regulation Of The Bombyx Mori A3 Cytoplasmic Actin Promoter In Insect And Mammalian Cells, Karoly Fatyol

Loma Linda University Electronic Theses, Dissertations & Projects

In this study we have analyzed the activity of the silkworm A3 cytoplasmic actin promoter in homologous insect and heterologous mammalian expression systems. To study the expression of the A3 actin promoter in insect cells we have isolated and characterized a stably transformed Bombyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. Integration of the transfected plasmids into the host genome was demonstrated by Southern and in situhybridizations. By using this cell line we have identified alternative transcriptional initiation sites for the A3 …